We developed an approach to construct and probe protein microarrays on a genome-wide scale to characterize host antibody responses against more than 25 medically important infectious microorganisms. The lab has made 35,000 plasmids, printed the encoded proteins on 25,000 microarrays and probed them with 12,000 human serum specimens. The individual proteins printed on these arrays capture antibodies present in the serum of infected individuals, the amount of captured antibody can be quantified using fluorescent secondary antibody and antibody profiles that result after infection determined. Here we propose to apply this approach toward understanding the basis of naturally-acquired humoral immunity (NAI) to clinical malaria, to identify antibody biomarkers associated with protection from P. falciparum infection, and to discover novel malaria vaccine antigen candidates. People living in areas of intense Plasmodium falciparum (Pf) transmission develop resistance to the symptoms of malaria after repeated exposures to Pf parasite infection through childhood and adolescence. Probing a protein microarray containing 2,320 Pf proteins with sera collected from Pf-exposed Malian children and adults indicated that long-lived antibody responses are only acquired after years of repeated Pf infections and protective antibodies were identified against 49 specific Pf antigens.(2) Here we aim to extend these preliminary findings with a more comprehensive immuno-epidemiological analysis in Mali. Four thousand plasma samples which have already been collected from three NIAID-supported studies in Mali will be probed and analyzed. The longitudinal nature of these studies allows for antibody profiling at strategic time points before, during and after Pf infection and thus permits a prospective analysis of the relationship between antibody profiles and protection from malaria. We will also investigate the P. falciparum-specific antibody profile of infants and children aged 3 months to 5 years as they transition from maternally-derived antibodies to naturally-acquired antibodies through P. falciparum exposure. Finally, in a cross-sectional analysis we will investigate the isotype profile and antigen specificity of Pf-specific antibodies in the Fulani and Dogon people in Mali, distinct ethnic groups which have well-documented differential susceptibility to malaria. This proposal also takes advantage of recent funding from the NIAID Genome Sequencing Center (in collaboration with the J. Craig Venter Institute) to perform genotyping of targeted genomic regions of 700 individuals in one of the cohort studies as well as genome-wide transcription profiling (by RNA-seq.) of the same individuals before, during and after P. falciparum infection. This will provide a unique opportunity to examine prospectively the relationship between genetic polymorphisms, leukocyte transcription profiles and P. falciparum-specific antibody responses. PUBLIC HEALTH RELEVANCE: People living in areas where malaria is endemic get infected repeatedly with malaria and develop an elaborate array of antibodies against hundreds of parasite antigens. Eventually after many years of continuous exposure this array of antibodies is strong and diverse enough to confer protection against the symptoms of the disease, but the identity of the antibodies responsible for protection is unknown. Using our protein microarray approach we can examine all the individual antibody responses in the serum of infected people, determine which specific ones are responsible for conferring protection, and then test these antigens in subunit vaccines.